Culturing Anaplasma

ABSTRACT

The invention provides mammalian cells stably infected with  Anaplasma  species, as well as materials and methods related to propagating  Anaplasma  species in mammalian cells and isolating such  Anaplasma  species from mammalian cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. §371and claims benefit under 35 U.S.C. §119(a) of International ApplicationNo. PCT/US2003/025208 having an International Filing Date of Aug. 12,2003, which claims the benefit of priority under 35 U.S.C. §119(e) ofU.S. Application No. 60/403,261 having a filing date of Aug. 14, 2002.

BACKGROUND

1. Technical Field

The invention relates to methods and materials involved in culturingAnaplasma species.

2. Background Information

Anaplasma phagocytophilum and Anaplasma marginale are obligateintracellular, tick-borne rickettsial pathogens of humans and animals inNorth America, Europe, Australia, and Africa.

A. phagocytophilum (formerly known as “HGE agent,” Ehrlichia equi, or E.phagocytophila) causes disease in humans, horses, small and largeruminants, dogs, and cats. A. phagocytophilum infections produce anacute, febrile illness accompanied by appearance of the microbes inwhite blood cells (specifically neutrophil granulocytes, as well astheir precursors in the bone marrow), a reduction in the number of allblood cell types (“pancytopenia”), nausea, and confusion. Death occursin about 5% of human patients if not treated promptly with tetracyclineantibiotics. Diagnosis during the acute stage is difficult due to theabsence of significant amounts of specific antibodies at that time, anda vaccine is not yet available. Recently, the causative agent has beenisolated in cell lines of both human (the promyelocytic human leukemiacell line HL-60) and vector tick (the Ixodes scapularis cell lines ISE6and IDE8) origin (See Munderloh et al., 1996, J. Clin. Microbiol.,34:664-670; and Munderloh et al., 1999, J. Clin. Microbiol.,37:2518-2524).

A. marginale is only known to infect red blood cells in ruminants,specifically cattle, often being referred to in the literature as anobligate intraerythrocytic pathogen. The disease is characterized byanemia, weakness, loss of milk production, retarded growth, abortion,and, in severe cases, death. The continuous propagation of this microbein tick cell culture using the I. scapularis cell line IDE8 has beenreported (Munderloh et al., 1996, J. Med. Ent., 33:656-664). Despitetheir availability, tick cell-based cell culture systems have provendifficult to use for many research laboratories in industry andacademia.

SUMMARY

The invention provides methods and materials related to propagatingAnaplasma species in mammalian cells. Specifically, the inventionprovides for mammalian nucleated cells and mammalian adherent cells thatare stably infected with an Anaplasma species, as well as methods andmaterials for making such mammalian cells. In addition, the inventionprovides methods and materials for (1) propagating various Anaplasmaspecies in stably infected mammalian cells and (2) obtaining Anaplasmaspecies from stably infected mammalian cells. The invention is based onthe discovery that some mammalian cells such as endothelial cells andVero cells can be stably infected with Anaplasma species, for example A.marginale and A. phagocytophilum. As such, these mammalian cells can beused as vehicles for propagating Anaplasma species in vitro. Such aculture system can allow Anaplasma to be clonally selected for geneticanalysis, and can provide a ready source of Anaplasma that can be usedas antigen for the production of anaplasmosis diagnostics andanaplasmosis treatment materials (e.g., Anaplasma vaccines).

In one aspect, the invention provides an isolated mammalian cell stablyinfected with an Anaplasma species such as A. marginale, A. centrale, A.bovis, A. ovis, and A. platys, wherein the mammalian cell is a nucleatedcell. For example, the nucleated mammalian cell can be infected with A.marginale, or with A. centrale, or with A. bovis, or with A. ovis, orwith A. platys.

In another aspect, the invention provides an isolated mammalian cellstably infected with an Anaplasma species such as A. marginale, A.phagocytophilum, A. centrale, A. bovis, A. ovis, and A. platys, whereinthe mammalian cell is an adherent cell. For example, the adherentmammalian cell can be infected with A. marginale, or with A.phagocytophilum, or with A. centrale, or with A. bovis, or with A. ovis,or with A. platys.

Typically, the mammalian cell is an endothelial cell. Representativeendothelial cells include, without limitation, a bovine cornealendothelial cell, a rhesus monkey microvascular endothelial cell, ahuman umbilical vascular endothelial cell, and a human microvascularendothelial cell.

In an embodiment, the invention provides an isolated mammalian cellstably infected with Anaplasma marginale, wherein the mammalian cell isa nucleated cell. In another embodiment, the invention provides anisolated mammalian cell stably infected with Anaplasma phagocytophilum,wherein the mammalian cell is an adherent cell.

In one aspect, the invention provides a method of making a mammaliancell that is stably infected with an Anaplasma species such as A.marginale, A. centrale, A. bovis, A. ovis, and A. platys. Such a methodincludes contacting a nucleated mammalian cell with the Anaplasmaspecies to produce a mammalian cell stably infected with the Anaplasmaspecies. For example, the nucleated mammalian cell can be contacted withA. marginale, or with A. centrale, or with A. bovis, or with A. ovis, orwith A. platys.

In another aspect, the invention provides a method of malting amammalian cell that is stably infected with an Anaplasma species such asA. marginale, A. phagocytophilum, A. centrale, A. bovis, A. ovis, and A.platys. Such a method includes contacting a mammalian adherent cell withthe Anaplasma species to produce a mammalian cell stably infected withthe Anaplasma species. For example, the adherent mammalian cell can becontacted with A. marginale, or with A. phagocytophilum, or with A.centrale, or with A. bovis, or with A. ovis, or with A. platys.

In an embodiment, the invention provides a method of making a mammaliancell that is stably infected with Anaplasma marginale. Such a methodincludes contacting a nucleated mammalian cell with A. marginale toproduce a mammalian cell stably infected with A. marginale. In anotherembodiment, the invention provides a method of making a mammalian cellthat is stably infected with Anaplasma phagocytophilum. Such a methodincludes contacting a mammalian adherent cell with A. phagocytophilum toproduce a mammalian cell stably infected with A. phagocytophilum.

In still another aspect, the invention provides a method for propagatingan Anaplasma species such as A. marginale, A. centrale, A. bovis, A.ovis, and A. platys. Such a method includes contacting a nucleatedmammalian cell with the Anaplasma species to produce a mammalian cellstably infected with the Anaplasma species, and culturing the stablyinfected mammalian cell. For example, the nucleated mammalian cell canbe contacted with A. marginale, or with A. centrale, or with A. bovis,or with A. ovis, or with A. platys. Generally, the cell is contactedwith A. marginale. A. marginale can be obtained from tick cells (invitro or in vivo) or red blood cells.

In still another aspect, the invention provides a method for propagatingan Anaplasma species such as A. marginale, A. phagocytophilum, A.centrale, A. bovis, A. ovis, and A. platys. Such a method includescontacting a mammalian adherent cell with the Anaplasma species toproduce a mammalian cell stably infected with the Anaplasma species, andculturing the stably infected mammalian cell. For example, the adherentmammalian cell can be contacted with A. marginale, or with A.phagocytophilum, or with A. centrale, or with A. bovis, or with A. ovis,or with A. platys. Generally, the cell is contacted with A.phagocytophilum. A. phagocytophilum can be obtained from HL-60 cells orwhite blood cells.

Generally, the mammalian cell is an endothelial cell or a Vero cell.Representative mammalian cells include, without limitation, a bovinecorneal endothelial cell, a rhesus monkey microvascular endothelialcell, a human umbilical vascular endothelial cell, and a humanmicrovascular endothelial cell. Typically, the Anaplasma species can bepropagated in mammalian cells for at least 8 weeks (e.g., 10 weeks, 2months, 6 months, 9 months, 12 months, 18 months, 2 years, 5 years, or10 years).

In an embodiment, the invention provides a method for propagatingAnaplasma marginale. Such a method includes contacting a nucleatedmammalian cell with A marginale to produce a mammalian cell stablyinfected with A. marginale, and culturing the stably infected mammaliancell. In another embodiment, the invention provides a method forpropagating Anaplasma phagocytophilum. Such a method includes contactinga mammalian adherent cell with A. phagocytophilum to produce a mammaliancell stably infected with A. phagocytophilum, and culturing the stablyinfected mammalian cell.

In another aspect, the invention provides a method for obtaining anAnaplasma species such as A. marginale, A. centrale, A. bovis, A. ovis,and A. platys. Such a method includes culturing a nucleated mammaliancell stably infected with the Anaplasma species, and isolating theAnaplasma species from the mammalian cell. For example, the nucleatedmammalian cell can be infected with A. marginale, or with A. centrale,or with A. bovis, or with A. ovis, or with A. platys. Generally, thecell is infected with A. marginale. In an embodiment, the Anaplasmaspecies is an attenuated Anaplasma species.

In another aspect, the invention provides a method for obtaining anAnaplasma species such as A. marginale, A. phagocytophilum, A. centrale,A. bovis, A. ovis, and A. platys. Such a method includes culturing amammalian adherent cell stably infected with the Anaplasma species, andisolating the Anaplasma species from the mammalian cell. For example,the adherent mammalian cell can be infected with A. marginale, or withA. phagocytophilum, or with A. centrale, or with A. bovis, or with A.ovis, or with A. platys. Generally, the cell is infected with A.phagocytophilum. In an embodiment, the Anaplasma species is anattenuated Anaplasma species.

In one embodiment, the invention provides for a method for obtainingAnaplasma marginale. Such a method includes culturing a nucleatedmammalian cell stably infected with A. marginale, and isolating A.marginale from the mammalian cell. In another embodiment, the inventionprovides a method for obtaining Anaplasma phagocytophilum. Such a methodincludes culturing a mammalian adherent cell stably infected with A.phagocytophilum, and isolating A. phagocytophilum from the mammaliancell.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The invention provides methods and materials related to propagatingAnaplasma species in mammalian cells. Specifically, the inventionprovides for mammalian nucleated cells and mammalian adherent cells thatare stably infected with an Anaplasma species, as well as methods andmaterials for malting such mammalian cells. In addition, the inventionprovides methods and materials for obtaining various Anaplasma species,e.g. A. marginale and A. phagocytophilum. The invention is based on thediscovery that some mammalian cells can be stably infected withAnaplasma species making them useful as vehicles for propagatingAnaplasma species in vitro.

Mammalian Host Cells Stably Infected with Anaplasma Species

The invention provides isolated mammalian cells that have been stablyinfected with Anaplasma. The term “isolated” as used herein withreference to a mammalian cell refers to a mammalian cell that has beenseparated from other cell types with which it is normally found innature. Isolated mammalian cells include, without limitation,untransformed or transformed (e.g. immortalized) mammalian cellsisolated from mammalian tissues. Other examples of isolated mammaliancells include, without limitation, hepatocytes isolated from liver andendothelial cells isolated from umbilical cord.

As used herein, the term “stably infected” refers to a host cell that(1) has been infected with an organism, i.e. an infective agent such asA. marginale, and (2) allows for multiplication of the infective agentwithin the host cell until the host cell lyses leading to release ofinfective agents from the host cell. When a culture of stably infectedcells lyses, infective agents in the culture lysate can be passaged to aculture of uninfected cells. For example, a culture lysate containinginfective agents can be used as an inoculum to infect other cells of thesame or different cell line to generate a second culture of stablyinfected cells. In this way, an Anaplasma species can be propagated forat least 10 passages through a host cell line without loss of theinfective agent. Anaplasma can be cultured in mammalian cells for weeks,months, or years. Loss of the infective agent can be monitored byexamining inclusions observed in infected cells as described in Blouinet al. (1993) Revue Elev Med vet Pays trop 46:49-56 for bovine turbinatecells and bovine pulmonary aorta endothelial cells. Thus, a stablyinfected cell such as a stably infected Vero cell or endothelial cellcan be used as a vehicle for propagating the Anaplasma species.

Mammalian host cells stably infected with an Anaplasma species include,without limitation, (1) mammalian nucleated cells that have been stablyinfected with A. marginale, and (2) mammalian adherent cells that havebeen stably infected with A. phagocytophilum. As used herein, anadherent cell refers to a cell that attaches firmly to a culturesubstrate (i.e., a plastic surface) in vitro. Adherent cells must bephysically detached from the substrate (e.g., scraped off with asilicone rubber-coated blade or treated with an enzyme solution (e.g.,trypsin). Examples of adherent cell types include endothelial cells,macrophages and other cells derived from the monocytic white blood celllineage, fibroblasts, and epithelial cells. Adherent cells are generallyderived from solid organs and white blood cells in the monocytic lineage(e.g., macrophages, histiocytes, and Kupffer cells). Non-adherent cells(e.g., red blood cells, HL-60 cells, or bone marrow cells other thanthose of the monocytic lineage) may settle loosely onto the substrateand can be resuspended by agitating the medium. Scraping or enzymetreatment is not required to resuspend non-adherent cells.

Endothelial cells are particularly useful hosts cells for achievingstable infection with Anaplasma species. Examples of useful host cellsinclude bovine corneal endothelial cells (e.g., BCE C/D-1b cells; ATCCCRL-2048), rhesus monkey microvascular endothelial cells (e.g., RF/6Acells; ATCC CRL-1780), human umbilical vascular endothelial cells(HUV-EC-C; ATCC CRL-1730), human umbilical vein endothelial cells(HUVEC-12; ATCC CRL-2480), and African green monkey kidney cells (Verocells; ATCC CCL-81). Examples of Anaplasma species include A. marginaleand A. phagocytophilum. Other examples of Anaplasma species include,without limitation, A. centrale, A. bovis, A. ovis, and A. platys.

Mammalian host cells can be stably infected with Anaplasma species usingmethods known in the art. In one embodiment, Anaplasma can be added tothe host cell culture, and the culture incubated under conditionstypically used for culturing the particular host cell type. For example,a suitable amount (e.g. 1 mL) of a lysed cell suspension derived from atick cell culture (e.g. ISE6 cells) in which 70% or more of the tickcells are stably infected with A. phagocytophilum can be added to aculture of the suitable mammalian host cell, e.g. an endothelial cellsuch as the RF/6A cell. The inoculated mammalian cell culture then canbe grown under appropriate conditions, e.g., in L15B300 mediumsupplemented as described herein in tightly closed flasks incubated at37° C. The resulting culture can be grown in this manner until celllysis occurs. Before host cell lysis, Anaplasma can be observed asinclusion bodies in the cytoplasm of host cells. Once cell lysis occurs,the culture lysate can be used to inoculate uninfected cells asdescribed above.

Detecting Anaplasma

Anaplasma can be detected by several methods including, withoutlimitation, microscopic analysis of live cultures, as well ashistochemical, immunocytological, and PCR analyses. Such methods areparticularly useful for determining whether host cells such as mammalianendothelial cells have been stably infected with Anaplasma.

Histochemical analysis can be performed on infected cells stained withGiemsa stain. Infected cells then can be detected by observing thepresence of Anaplasma inclusions in the cytoplasm, similar to those seenin vivo in neutrophilic granulocytes. Inclusions in heavily infectedcells can completely fill the cytoplasm, and cause the cell to becomedistended. For example, a sample of endothelial cells infected with A.marginale and passaged for 9 months can be processed using thehistochemical methods described herein. The presence of Giemsa-stainedinclusions in the cytoplasms of endothelial cells indicates the presenceof A. marginale in these cells.

Alternatively, Anaplasma can be detected by immunocytological methodswith specific A. marginale or A. phagocytophilum antibodies that arefluorescently labeled. For example, a sample of endothelial cellsinfected with A. phagocytophilum and passaged for 6 months can beexposed to fluorescently labeled antibodies specific for A. marginale orA. phagocytophilum. The presence of Anaplasma in the cell sample isindicated by the presence of immunofluorescence after the cell samplehas been washed with appropriate buffers to eliminate non-specificbinding of the Anaplasma antibody.

Anaplasma also can be detected by PCR using oligonucleotide primersdesigned specifically to amplify only Anaplasma nucleic acid. PCR can beperformed on nucleic acid isolated from Anaplasma-infected cells.Primers can be designed based upon nucleic acid sequences found in theAnaplasma genome. See, for example, The Ehrlichia Research Laboratory,Ohio State University Health Sciences Center, College of VeterinaryMedicine, Columbus, Ohio, and Animal Disease Research Unit-USDA-ARS,Dept. of Veterinary Microbiology and Pathology and the College ofVeterinary Medicine, Washington State University, Pullman, Wash.).Primers that amplify an Anaplasma nucleic acid molecule can be designedusing, for example, a computer program such as OLIGO (Molecular BiologyInsights, Inc., Cascade, Colo.). Important features when designingoligonucleotides to be used as amplification primers include, but arenot limited to, an appropriate size amplification product to facilitatedetection (e.g., by electrophoresis), similar melting temperatures forthe members of a pair of primers, and the length of each primer (i.e.,the primers need to be long enough to anneal with sequence specificityand to initiate synthesis but not so long that fidelity is reducedduring oligonucleotide synthesis). Typically, oligonucleotide primersare 8 to 50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength).

Use of Mammalian Cells Stably Infected with Anaplasma

Mammalian host cells stably infected with an Anaplasma species can beused for a variety of purposes including propagation of selectedAnaplasma species. For example, mammalian host cells such as RF/6A cellsthat are stably infected with A. phagocytophilum can be a source ofinfectious A. phagocytophilum since the infected RF/6A cells allow forthe intracellular multiplication of infectious A. phagocytophilum.Subsequent host cell lysis results in release of infectious A.phagocytophilum into the culture medium. The resulting culture lysatecan be used as an inoculum for infecting uninfected cells. Using suchprocedures and passaging through numerous cell cultures, A.phagocytophilum can be propagated in vitro for four months or longer.

Infectious A. phagocytophilum propagated in this manner can be used toinfect other host cell types for pathogenesis studies. Stably infectedhost cells can be cultured for various lengths of time before cell lysisnecessitates passage to an uninfected cell or cell culture. The lengthof time between passages can vary depending on the particular cultureconditions, host cell type, and Anaplasma species, and can be from lessthan one week to about one month or more. The length of time betweenpassages for A. phagocytophilum grown in RF/6A cells under conditionsdescribed herein, for example, is one week or less.

Mammalian host cells stably infected with an Anaplasma species also canbe used to develop materials for the treatment of anaplasmosis andanaplasmosis-related diseases, e.g., ehrlichioses such as humangranulocytic ehrlichiosis (HGE), and determining the efficacy of suchanaplasmosis treatment materials. For example, mammalian host cellsstably infected with an Anaplasma species can be used as a source ofAnaplasma. Anaplasma isolated from host cell cultures using methodsdescribed herein can be used for developing anaplasmosis treatmentmaterials such as agents for treating, controlling, or preventinganaplasmosis (e.g., Anaplasma vaccines), as well as growth promoters,feed additives, pharmaceuticals, nutraceuticals, antibiotics, andantimicrobial agents. Anaplasmosis treatment material formulations caninclude vaccine formulations containing whole microorganisms or antigenpreparations containing portions of the whole microorganism. The use ofwhole microorganisms in a treatment material formulation typicallyinvolves attenuation. The term “attenuated” as used herein refers to areduction in the virulence of an infective agent such as A. marginale orA. phagocytophilum. Attenuation can be performed using various methodsincluding, without limitation, exposure to heat (e.g., heat-killing),and chemical inactivation (e.g., treatment with β-propiolactone).Alternatively, an infective agent such as an Anaplasma species can becultivated in vitro until the organism has lost the ability to infecthost animals or humans. Treatment material formulations containingattenuated organisms can be prepared in accordance with methods standardin the art.

Attenuated whole Anaplasma organisms and/or Anaplasma antigenpreparations containing portions of whole Anaplasma can be combined withphysiologically acceptable carriers. Such physiologically acceptablecarriers include, without limitation, buffered salt solutions, phosphatebuffered saline, and cell culture medium. In addition, treatmentmaterial formulations also can include adjuvants, such as alum, ISCOMs,complete Freund's adjuvant, incomplete Freund's adjuvant, and saponin.

Anaplasmosis treatment materials can be administered by a variety ofroutes including, without limitation, intravenous, intraperitoneal,intramuscular, subcutaneous, intrathecal, and intradermal injection, byoral administration, by inhalation, or by gradual perfusion over time.For example, a solution preparation containing heat-killed Anaplasma canbe given to a host by intramuscular injection. It is noted that theduration of treatment with any of the materials described herein can beany length of time from as short as one day to as long as a lifetime(e.g., many years). For example, an anaplasmosis treatment material canbe administered once a year over a period of ten years. It is also notedthat the frequency of treatment can be variable. For example, ananaplasmosis treatment material can be administered once daily for 20days, then twice monthly for 6 months, and then once yearlyindefinitely. Typically, an anaplasmosis treatment material isadministered at a frequency that induces and maintains a protectiveeffect (i.e., a protective immune response to infection by an Anaplasmaspecies) in the treated mammal. Treatment materials also can ameliorateand/or prevent the development of symptoms associated with anaplasmosis(e.g., anemia, weakness, and retarded growth).

Any method can be used to determine if a particular immune response isinduced. For example, antibody responses against particular antigens canbe determined using immunological assays (e.g., ELISA) such as thosedescribed herein. In addition, clinical methods that can assess theseverity of a particular disease state (e.g., Anaplasma infection) canbe used to determine if a desired immune response is induced.

Antigen preparations are also useful for diagnostic assays, such asELISA, solid phase immunoassays, complement fixation tests, delayed-typehypersensitivity response assays and the like. To obtain an Anaplasmaspecies likely to include antigens that react with antibodies producedafter mammalian infection, antigens can be prepared from Anaplasmaspecies obtained from stably infected mammalian endothelial cells.

Anaplasma organisms can be used to determine the efficacy of ananaplasmosis treatment material. Once given a particular anaplasmosistreatment material, a mammal can be challenged with Anaplasma organisms.Once challenged, the mammal is assessed to determine the presence orabsence of an Anaplasma infection. For example, a cow given an Anaplasmavaccine can be challenged with A. marginale and then assessed todetermine the presence or absence of an Anaplasma infection. Determiningthe presence or absence of an Anaplasma infection in a treated mammalprovides an indication of the efficacy of that treatment material. Forexample, if a cow treated with an Anaplasma vaccine develops anAnaplasma infection after being challenged with A. marginale, then thatAnaplasma vaccine may not be effective in protecting that cow againstAnaplasma infection. Alternatively, if a cow treated with an Anaplasmavaccine does not develop an Anaplasma infection after being challengedwith Anaplasma, then that Anaplasma vaccine likely is effective inprotecting that cow against Anaplasma infection.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Host Cell Lines

Tick cell line ISE6, from embryos of the black-legged tick, Ixodesscapularis, was used for propagation of both A. marginale and A.phagocytophilum (Munderloh et al., 1996, J. Med. Entomol., 33:656-64;Munderloh et al., 1996, J. Clin. Microbiol., 34:664-670; and Munderlohet al., 1999, J. Clin. Microbiol., 37:2518-2524). Uninfected cells weregrown in L15B300 with 5% tryptose phosphate broth (Difco Laboratories,Detroit, Mich., USA), 5% heat-inactivated fetal bovine serum (FBS,Harlan, Indianapolis, Ind., USA), and 0.1% bovine lipoproteinconcentrate (ICN, Irvine, Calif., USA), pH 7.2. Medium for infectedcultures was additionally supplemented with 25 mM HEPES[N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)] and 0.25%NaHCO₃, and the pH was adjusted to 7.5-7.7. All cultures were maintainedat 34° C. Anaplasma were passaged by transferring 1/50th of an infectedcell suspension to a new flask of tick cells every 2 weeks.

The mammalian cells employed in this study were endothelial lines RF/6A(American Type Culture Collection, Manasssas, Va., USA; ATCC CRL-1780)from the retina choroid of a normal fetal rhesus (Macaca mulatta), BCEC/D1-b, an adult bovine corneal endothelial cell line that is free ofbovine viral diarrhea/mucosal disease virus (ATCC CRL-2048), the humanmicrovascular cell line HMEC-1 (Ades et al., 1992, J. Invest. Dermatol.,99:683-690), and primary human skin microvascular endothelial cells(MVEC; VEC Technologies, Inc. Rensselaer, N.Y., USA). The humanpromyelocytic leukemia cell line HL-60 (ATCC CCL-240) was used topropagate A. phagocytophilum as described earlier (Goodman et al., 1996,N. Engl. J. Med., 334:209-215). Rhesus and bovine endothelial cells weregrown in closed flasks in L15B300 supplemented as for infected ISE6cells, except that 10% FBS and 50 mM HEPES was used. HL-60 cells weremaintained in RPMI1640 (Bio Whittaker, Walkersville, Md., USA); HMEC-1cells were grown in MCDB 131 (Mediatech, Herndon, Va., USA) with 0.5μg/ml cortisone; and MVEC were propgated in complete MCDB-131 from VECTechnologies, all with 10% FBS (HyClone, Logan, Utah, USA) and in a 5%CO₂ atmosphere. Mammalian cell cultures were kept at 37° C. Endothelialcells were detached using trypsin (Gibco, Grand Island, N.Y., USA), anddiluted 4-fold once a week for subculturing.

Example 2 Infection of Endothelial Cells with A. marginale and A.phagocytophilum

For A. marginale, the Virginia isolate Am291 in its 36th passage in ISE6cells was used as the primary inoculum. For A. phagocytophilum, ISE6cells infected with the 24th passage of the isolate HGE2 was used.Initially, 0.5 ml of an Anaplasma culture in which 80% or more of thetick cells were infected and releasing bacteria due to cell lysis, wasadded to a 25 cm² flask with a confluent endothelial cell layer, linesRF/6A, BCE C/D-1b, and HMEC-1, or primary MVEC. For some experimentswith HGE2, endothelial cells were alternatively inoculated with 50 μl(equivalent to 5×10³ infected cells) of A. phagocytophilum from HL-60cells at various passage levels of 23 and higher. The organisms wereharvested from infected HL-60 cells by mechanical rupture as a hostcell-free suspension as described, and added directly to recipientcultures in 5 ml medium per 25 cm² flask, or 0.5 ml medium per well of a24-well plate. Cultures were incubated in their respective media andatmospheric conditions at 37° C. Cultures were monitored daily by phasecontrast microscopy. For continuous passage in endothelial cells,infected cell layers were scraped off the growth surface, the suspensionrepeatedly pipetted to disrupt cell clumps, and a portion transferred toa fresh, confluent cell layer.

Example 3 Light Microscopy

For Giemsa-staining or immunofluorescence assays (IFA), 25 cm2 celllayers were rinsed once with phosphate buffered saline, pH 7.5 withoutCa²⁺ and Mg²⁺ (PBS), and detached from the substrate by trypsinization.Cells were resuspended in growth medium, and 10⁴ cell aliquots spun ontomicroscope slides using a Cytospin (Shandon Southern Instruments,Sewickley, Pa., USA) centrifuge at 60×g for 5 min. Slides were fixed inabsolute methanol for 5 min, air-dried briefly, and immersed in abuffered (pH 6.8) solution of 4% Giemsa's stain (Karyomax, Gibco, GrandIsland, N.Y., USA) for 30 min at 37° C.

For IFA, fixed, air dried cell spots were overlaid with primary antibodydiluted as outlined below, and incubated in a humid atmosphere at roomtemperature for 1 hr. Slides were rinsed in PBS, and immersed in PBSwith 10% bovine serum albumin (BSA; Serologicals Corporation, Norcross,Ga., USA) for 10 min. They were then dipped in distilled water, rinsedin PBS, and the cells covered with fluorescein isothiocyanate(FITC)-labeled IgG of the appropriate species specificity for 1 hr atroom temperature in a humid chamber. Finally, the slides were rinsedwith PBS, counterstained in Evans' Blue (0.005% in PBS) and covered withantifade mounting medium (Vector Laboratories, Burlingame, Calif., USA).

Primary antisera used were a bovine hyperimmune serum to A. marginaleinitial bodies harvested from erythrocytes (kindly provided by Dr.Katherine M. Kocan, Oklahoma State University, Stillwater; Munderloh etal., 1996, J. Med. Eutonol., 33:656-64), and a mouse monoclonal antibodyagainst MSP2 of A. phagocytophilum (a generous gift from Dr. Russell C.Johnson, University of Minnesota; Ravyn et al., 1999, Am. J. Trop. Med.Hyg., 61:171-176). Bovine anti-A. marginale serum was diluted 1:200, andthe monoclonal antibody was diluted 1:10,000.

Slides were viewed and photographed under oil immersion at 100×magnification using a Nikon Eclipse E400 microscope, fitted forepifluorescence and equipped with a Nikon DXM1200 digital camera.

Example 4 Electron Microscopy

Cultures estimated by phase contrast microscopy to be 50% or moreinfected, were scraped off their substrate, approximately 5×10⁴ cellswere pipetted into 1 ml of Ito's modified fixative (Kurtti et al., 1994,Can. J. Zool., 72:977-994), and incubated on ice for 1 hr. Fixed cellswere collected by centrifugation at 275×g for 5 min, and resuspended in1.5 ml fresh fixative. Cell pellets were postfixed in osmium tetroxideand dehydrated in graded changes of an ascending alcohol series. Thepellet was embedded in Spurr's epoxy resin and thin sections werestained with methanolic uranyl acetate and Reynold's lead citrate.

Example 5 Polymerase Chain Reaction (PCR)

Table 1 lists the primers used in this study. To verify the identity ofA. marginale or A. phagocytophilum from endothelial cell culture, foursets of primers were employed; one directed at the 16S rDNA of thegenera Ehrlichia and Anaplasma (PER1, PER2), yielding a 451 bp product(Goodman et al., 1996, N. Engl. J. Med., 334:209-215), two that bind tothe msp2 (p44) gene of A. phagocytophilum (p44-1, p44-2; and p3708,p4257) resulting in a 1,279 bp and a 541 bp product, respectively (Ijdoet al., 1998, Infect. Immun., 66:3264-3269; Zhi et al., 1999, J. Biol.Chem., 274:17828-17836), and one that is specific for the conservedregion of the A. marginale msp1β gene (AL34S, BAP-2; Barbet and Allred,1991, Infect. Immun., 59:971-976; Stich et al., 1993, J. Med. Entomol.,30:789-794) and mediates amplification of a 407 bp target. In controlreactions, sterile water was substituted for DNA. Endothelialcell-culture derived Anaplasma were separated from host cells,solubilized in lysis buffer, and DNA extracted using the PureGene kit(Gentra Systems, Minneapolis, Minn., USA) as described (Goodman et al.,1999, J. Clin. Invest., 103:407-412). DNA was dissolved in sterile water(500 μl for each DNA pellet harvested from one 25 cm² culture), andstored at −20° C. Two μl of DNA was used in each 50 μl reaction mixturecontaining 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl₂, 200 μM eachof deoxynucleotides, 0.5 μM of each oligonucleotide primer, and 1.25units of Taq DNA polymerase (Promega, Madison, Wis.). DNA was initiallydenatured for 3 min at 95° C., and then amplified in a Robocycler(Stratagene, La Jolla, Calif., USA) during 40 cycles consisting of adenaturation step of 30 sec at 94° C., annealing for 30 sec at 45° C.,and elongation for 45 sec at 72° C., with a final elongation step of 5min. DNA extracted from tick cells cultures (for A. marginale, Munderlohet al., 1996 J. Med. Entomol., 33:656-64) or HL-60 cell cultures (for A.phagocytophilum, Goodman et al., 1996, N. Engl. J. Med., 334:209-215)was used as a positive control, and reactions mixtures containing waterinstead of DNA served as negative controls. PCR products were separatedby electrophoresis through 1% agarose gels in 0.5× Tris-Borate-EDTAbuffer, and visualized by ethidium bromide staining and ultraviolettransillumination.

All primer pairs resulted in amplification of the correct size productof only their target DNA.

TABLE 1 Oligonucleotides Primer Designation, Specificity NucleotideSequence (5′ → 3′) and Target (SEQ ID NO:) Reference p44-1 Anaplasmaphagocytophilum AGC GTA ATG ATG TCT ATG GC (1) a p44-2 p44 (insp2) ACCTAA CAC CAA ATT CCC (2) a p3708 Anaplasma phagocytophilum GCT AAG GAGTTA GCT TAT GAT (3) b p4257 p44 (msp2) AAG AAG ATC ATA ACA AGC ATT (4) bPER1 Anaplasma and Ehrlichia-wide TTT ATC GCT ATT AGA TGA GCC TAT G (5)c PER2 16S rDNA CTC TAC ACT AGG AAT TCC GCT AT (6) c BAP-2 Anaplasmamarginale GTA TGG CAC GTA GTC TTG GGA TCA (7) d AL34S msp1β CAG CAG CAGCAA GAC CTT CA (8) e a Ijdo et al., 1998, Infect. Immun., 66:3264-9; bZhi et al., 1999, J. Biol. Chem., 274:17828-36; c Goodman et al., 1996,N. Eng. J. Med., 334:209-15; d Barbet and Allred, 1991, Infect. Immun.,59:971-6; e Stich et al., 1993, J. Med. Entomol., 30:789-94.

Example 6 Growth of Anaplasma in Endothelial Cell Lines

Both A. marginale and A. phagoytophilum that had been continuouslypropagated in I. scapularis cell line ISE6, when inoculated onto RF/6Arhesus, BCE C/D-1b bovine or HMEC-1 human endothelial cell layers,invaded these cells and replicated inside intracellular inclusions.After the initial inoculation with infected tick cells, inclusions ofeither Anaplasma sp. could be detected by phase contrast microscopy inRF/6A cells within several days. The first passage of A. phagocytophilumfrom ISE6 cells caused RF/6A lysis within 2 weeks, but subsequentreplication was much faster, and by the 10th passage, cultures wereroutinely subcultured by diluting 100- or 200-fold every 5-7 days, or1,000-fold every 10 days. A. marginale grew more slowly in RF/6A than A.phagocytophilum. The initial passage from tick cells caused infection in100% of cells in 10 days, but subsequent subcultures were made with a10-fold dilution of infected cells every 2 weeks. These growth rates inRF/6A cells have remained stable for A. marginale and A.phagocytophilum.

Infection dynamics of both Anaplasma spp. in BCE C/D-1b bovineendothelial cells were different from those in rhesus cells. The initialtransfer of A. marginale from ISE6 tick cells resulted in 70% infectionwithin 30 days, when the cells were further passaged to fresh BCE C/D-1bat a 1:5 dilution. The next 1:5 passage was carried out with 90%infected cells 28 days later, but this culture failed to becomeestablished, and was subsequently discarded. Similarly, A.phagocytophilum could be transferred three times in BCE C/D-1b cellswithin a time-span of 46 days, growing to infect 80-90% of the cellseach time, and then stopped replicating further, and was alsodiscontinued. When BCE C/D-1b cells were inoculated with Anaplasmaderived from RF/6A cells, they behaved like those derived from tickcells, and the bacteria stopped growing after 3 or 4 transfers. Thesecultures were not pursued further. In RF/6A and BCE C/D-1b, control ofthe medium pH at or above 7.5 was critical, and was achieved by doublingthe concentration of HEPES over that used in tick cells.

Only A. phagocytophilum replicated in HMEC-1 and MVEC cells. Bacteriataken from either HL-60 or RF/6A cultures readily invaded and multipliedin HMEC-1, while A. marginale transferred at the same time from RF/6Acells did not. ISE6 culture-grown A. phagocytophilum also infectedHMEC-1 cells, but took 10 or more days to become apparent by lightmicroscopy. Once established, it could then be passaged at the sameschedule as bacteria transferred from HL-60 cells to HMEC-1.

Example 7 Light Microscopic Features of Anaplasma in Endothelial Cells

The appearance of A. marginale and A. phagocytophilum was distinct atthe light microscopic level. Notably, A. marginale formed largeinclusions in RF/6A that appeared smooth and solid by phase contrastmicroscopy during the first few days. Individual bacteria, too tightlyjuxtaposed to each other to discern at first, subsequently condensed andbecame discrete, visible as a large number of tiny granules containedwithin a single inclusion. Eventually, the host cell ruptured andreleased bacteria that spread through the culture, causing completedestruction of infected monolayers. By contrast, A. phagocytophilumtended to form numerous smaller and very distinct inclusions (morulae)in each cell in which individual Anaplasmas were always distinguishable.RF/6A cells were infected with A. marginale or A. phagocytophilum, andHMEC-1 and MVEC cells were infected with A. phagocytophilum. A.marginale inclusions diffuse were, and large morulae were released froma ruptured host cells. Many well-defined morulae were evident in A.phagocytophilum-infected cells, with greater numbers accommodated in thelarger RF/6A host cell.

Both the bovine anti-A. marginale and the anti-A. phagocytophilumantibodies reacted with their target antigen in a specific manner.Either antibody preparation preferentially stained the periphery ofindividual bacteria or small groups of bacteria in a morula, resultingin a honeycombed or fish net-like pattern that was most noticeable inRF/6A and BCE C/D-1b cells, and less so in HMEC-1 and MVEC. In thelarger RF/6A and BCE C/D-1b cells, the Anaplasma inclusions were lesscompact than in HMEC-1 and MVEC cells, in which A. phagocytophilumformed primarily well defined and dense, rounded morulae.

Example 8 Electron Microscopy

Ultrastructural images confirmed many of the light microscopicobservations. A. marginale formed few but large inclusions per cell.These often contained bacteria that differed in degree of condensationand in shape. Many appeared to progress from being tightly packed withreticulate forms that abutted and conformed to each other like pieces ofa mosaic, to being loosely filled with condensed bacteria. A singleendosome had regions in which reticulated forms were grouped closelytogether, with dense forms loosely filling the remainder of theinclusion. By contrast, A. phagocytophilum morulae were small andnumerous, and most contained individual organisms of the samemorphologic type, i.e., either reticulated, or dense forms. Rarely, A.phagocytophilum morulae harbored both dense and reticulate bacteria. Themost unusual-looking bacteria were found in A. phagocytophilum.Sometimes rod-shaped, sometimes more rounded, they were electron denseand showed evidence of extensive invaginations and infolding ofmembranes. Reticulate forms present in the same morula indicate theseare not artifacts.

Example 9 Preparation of Cell Lysates Containing Anaplasma Antigens

Anaplasma antigens produced from Vero or endothelial cell cultures arerecognized by sera from infected animals and humans, and are useful forserologic diagnosis. Vero or endothelial cell cultures, infected andmaintained as described in Example 1, were cultured until 90% or more ofthe cells were infected with either A. marginale or A. phagocytophilum.The infected cells were scraped from the flask bottom and then shearedby repeated passage through a 27-gauge needle. Alternatively, the cellswere disrupted by sonication. The sheared cells were then centrifuged at400×g to settle large debris. The supernatant, containing Anaplasma, wasconcentrated by centrifugation at 2000×g for 20 minutes. The resultingpellet, containing Anaplasma, was then further purified by passagethrough a 30% renografin density gradient. Purified Anaplasma, collectedat the bottom of the gradient were resuspended in PBS, and then lysed bysonication. The protein concentration in the lysate was determined usingstandard techniques, and then adjusted to 5 μg/mL using 0.015 M Na₂CO₃in 0.035 M NaHCO₃, pH 9.6.

Example 10 Detection of Anaplasma Antigens in Cell Lysates

To detect Anaplasma antigens in cell lysate preparations, 100 μLaliquots of the diluted lysate prepared as described above were added toeach well of a 96-well, amine-binding microtiter plate. After 24 hoursat 4° C., the plates were washed three times with PBS, pH 7.2, andblocked with PBS containing 0.05% Tween 20 and 1% bovine serum albumin(BSA). After 1 hour at room temperature, the plates were washed threetimes with PBS, and 100 μL of sera serially diluted 10-fold in PBS wereadded to the wells. After one hour at room temperature, unbound serawere removed with three PBS washes, and then horseradishperoxidase-labeled antibodies recognizing IgG of the appropriate specieswere added to the wells. After one hour at room temperature unboundantibodies were removed with three PBS washes, and the bound antibodiesin the wells were treated with 0.4 mg/mL o-phenylenediamine phosphatefor 30 min. The reaction was stopped by addition of 1N H₂SO₄, andabsorbance at 490 nm was read using a plate reader or spectrophotometer.The absorbance values of test sera were compared with those from knownnegative sera. Test sera were considered positive when their absorbancevalues exceeded a value that corresponded to 3 standard deviations abovethe mean absorbance value for negative sera. A positive value istypically above 0.500. Using this method, Anaplasma antigens weredetected in lysates prepared from Vero and RF/6A cells infected with A.marginale. Therefore, Anaplasma antigens can be prepared from Vero orendothelial cell cultures.

Example 11 Detection of Anaplasma Antigens in Whole Cells

To detect Anaplasma antigens within confines of a mammalian host cell,Vero or endothelial cell cultures, infected and maintained as describedabove were cultured until 70% or more of the cells were infected witheither A. marginale or A. phagocytophilum. The growth medium was thenremoved from a flask and the cell layer was rinsed once with phosphatebuffered saline (PBS), pH 8. The rinsed cell layer was then flooded with3 mL 0.25% trypsin in PBS at pH 8. After one minute at room temperature,the trypsin solution was removed, and the culture was incubated at 37°C. until cells became rounded and detached from the growth substrate.The detached cells were suspended in about 5 mL of growth medium, andabout 5 μL of the cell suspension was deposited into wells of 18-wellslides. The cells were allowed to air dry over night, and were thenimmersed in 100% methanol or a mixture of 50% methanol and 50% acetone.After 10 minutes, the slides were briefly dried at room temperature, andstored desiccated at −20° C. To detect the presence of specificantibodies in patient sera, wells were sequentially incubated withserial dilutions of patient serum in PBS. After 60 minutes at 37° C.,the slides were rinsed three times in PBS. Labeled secondary antibodiesof the appropriate specificity (either anti-bovine or anti-human IgG)were then added to the wells. After 60 minutes at 37° C., the slideswere again rinsed three times in PBS. The rinsed slides were mounted tocoverslips using antifade mounting medium (e.g., PBS, 1% BSA (bovineserum albumin), 10% (w/v) triethylenediamine, and 10% glycerol, orVectashield from Vector Laboratories), and viewed under UV illuminationat 100× magnification using a microscope fitted for epifluorescence anda filter cube appropriate for the fluorescent label (either FITC orRhodamine). In this way, Anaplasma antigens were detected in Vero, BCEC/D 1-b and RF/6A cells by their bright green or red fluorescenceagainst the non-fluorescent background of the host cell.

Example 12 Vaccine Compositions Including Anaplasma Organisms Obtainedfrom Mammalian Cells

Vero or endothelial cells infected with A. marginale or A.phagocytophilum are cultured until phase contrast microscopy and/or cellsamples stained with Giemsa stain indicate that a majority (e.g., 90% ormore) of the cells are infected. Cultures are harvested, and Anaplasmamicroorganisms are purified through density gradient centrifugation asdescribed in Example 2. Purified Anaplasma microorganisms are countedusing a Petroff Hausser bacteria counter, and resuspended in PBS at adensity of approximately 5×10⁹ microorganisms/mL. Anaplasmamicroorganisms are inactivated with β-propiolactone as described byKocan et al. (2001, Vet. Parasitol., 102(1-2): 151-61).

Anaplasma microorganisms (2-5×10¹⁰ per dose) are mixed with adjuvant.The microorganisms are either absorbed onto aluminum hydroxide (alum) oremulsified with an oil-based adjuvant such as Adjuvant XtendIII(Novartis, Larchwood, Iowa). The microorganism/adjuvant mixture isadministered to a mammal over a specified period (e.g., two timessubcutaneously at four-week intervals, and once per year thereafter).The exact adjuvant and immunization protocol will vary depending on thespecies immunized and the treatment outcome desired.

These protocols provide improved vaccine compositions based on A.marginale or A. phagocytophilum antigens prepared from mammalian cells.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated mammalian cell stably infected with Anaplasma marginale,wherein said mammalian cell is a nucleated monkey cell.
 2. The isolatedmammalian cell of claim 1, wherein said mammalian monkey cell is anendothelial cell.
 3. The isolated mammalian cell of claim 2, whereinsaid cell is a rhesus monkey microvascular endothelial cell.
 4. A methodof making a mammalian cell that is stably infected with Anaplasmamarginale, said method comprising contacting a nucleated mammalianmonkey cell with said A. marginale to produce a mammalian cell stablyinfected with said A. marginale.
 5. A method for propagating Anaplasmamarginale, said method comprising contacting a nucleated mammalianmonkey cell with said A. marginale to produce a mammalian monkey cellstably infected with said A. marginale and culturing said stablyinfected mammalian monkey cell.
 6. The method of claim 5, wherein saidA. marginale is obtained from tick cells or red blood cells.
 7. Themethod of claim 5, wherein said mammalian monkey cell is an endothelialcell or a Vero cell.
 8. The method of claim 7, wherein said mammaliancell is a rhesus monkey microvascular endothelial cell.
 9. The method ofclaim 5, wherein said A. marginale is propagated for at least 8 weeks.10. A method for obtaining Anaplasma marginale, said method comprisingculturing a nucleated mammalian monkey cell stably infected with said A.marginale, and isolating said A. marginale from said mammalian monkeycell.
 11. The method of claim 10, wherein said A. marginale is culturedunder conditions in which said A. marginale is attenuated.